1. Introduction
2. Background of the Invention
2.1. Vaccines
2.2. Gonococcal Antigens
2.2.1. Gonococcal Protein I
2.3. Diagnostic Probes
3. Summary of the Invention
4. Description of Figures
5. Description of the Invention
5.1. Identification and Isolation and Sequence of the PIA Gene
5.2. Identification, Isolation and Sequence of the PIB Gene
5.3. Production of PIA, PIB or PIA/B Hybrids by Recombinant DNA Techniques
5.3.1. Preparation of a PIA Gene Cloning/Expression Vector
5.4. Identification and Purification of the PIA and PIB Gene Products
5.5. Preparation of PIA/B Hybrids
5.6. Production of PIA, PIB and Hybrid Proteins by Synthetic Techniques
5.7. Formulation of a Vaccine
6. Example I: PIA Gene Identification Isolation and Expression
6.1. General Procedures Used for Preparation of the Plasmids
6.1.1. Conditions for Restriction Enzyme
6.1.2. Restriction Enzyme Buffers
6.1.3. Identification of Relevant Restriction Fragments
6.1.4. Cloning of Fragments
6.1.5. Subcloning and Sequencing of Fragments
6.2. Gene Expression
7. Example 2: Identification, Isolation, Sequencing and Expression of PIB Gene and Construction and Expression of PIA/B Hybrids
7.1. Insertion of a Selectable Marker into the PI Gene
7.2. Cloning, Sequencing and Expression of the PIB Gene
7.3. Construction and Analysis of PIA/B Hybrid Strains
7.4. Discussion
8. Deposit of Microorganisms
Gonorrhea is at the present time one of the most widespread venereal disease worldwide, with several million cases occurring in the United States alone each year. The causative agent of the disease is the gonococcus Neisseria gonorrhoeae, a bacterium which has throughout its history developed resistance both to traditional antibiotic treatment, and to some extent to the bactericidal activity of normal human serum. The present inability to control the infection by traditional means has made the development of a vaccine which can effectively prevent the infection of utmost importance. The present invention provides a DNA sequence coding for a specific N. gonorrhoeae outer membrane protein; the cloned product of this particular DNA sequence provides a suitable basis for such a vaccine. The present invention also provides species-specific oligonucleotide sequences useful as diagnostic probes for the detection of gonorrheal infection.
2.1. Vaccines
The production of a protective immune response against any given infection agent in vertebrates depends initially on the provision of the appropriate stimulus to the host""s immune system. The infectious organism itself typically provides numerous stimulatory compounds, or antigens, by the very nature of its cell membrane composition, or by the metabolic products it releases in the host""s body. These substances, usually larger molecules such as proteins, lipopolysaccharides or glycoproteins, are recognized by the immune system as foreign, and provoke one or more different types of reaction from the host in an effort to remove or disable the invading organism. The antigen may cause production of sensitized lymphocytes (T-cells) which provide a cell-mediated immunity. Alternatively, an antigen may stimulate the synthesis and release of free antibody into the blood and other bodily fluids (humoral immunity). The development of the body""s protective immune response depends upon achieving a threshold level of stimulation of one or both of these systems.
A temporary immunity against infection can in many cases be provided by giving an individual preformed antibodies from another individual of the same or different species. This is known as passive immunity. One example of such immunity is the protection afforded to a fetus and newborn by placental transfer of maternal antibodies, as well as transfer of antibodies through milk. Another example is the pooled adult gamma globulin frequently used to prevent or modify the effects of exposure to measles, chicken pox, hepatitis, smallpox and tetanus. These acquired antibodies, however, are gradually utilized by interaction with the antigen or catabolized by the body, and thus the protection is eventually lost.
A more permanent form of protection is afforded by active immunization. Vaccination confers an active protective immunity by employing a harmless or nonvirulent form of the antigen (e.g., a killed or genetically altered bacterium, or an isolated glycoprotein from the cell wall) as a primary stimulus to the immune system. This provokes a rather slow response in antibody production which peaks and falls off. However, the body has been alerted to the existence of the antigen, and the next time exposure-occurs, presumably with the live, virulent organism, a secondary response, with a much more rapid and abundant production of antibodies is observed. This secondary response will typically be sufficient to prevent the microorganism from establishing itself sufficiently to be able to cause a full-blown infection.
A vaccine may take a variety of forms, some of which are more effective than others in conferring the protective effect desired. Historically many vaccines have been prepared by killing or inactivating the microorganism causing the disease of interest, and using the killed cells as the active immunogenic agent. Vaccines of this type have been used against typhoid, cholera and poliomyelitis (Salk vaccine). The problem with this type of vaccine is that the treatment required to kill the microorganisms, such as formaldehyde, may frequently alter or destroy the microbe""s useful antigens, and thus poor or incomplete immunity may be obtained.
An alternate form of vaccine is that which employs attenuated microorganisms. An attenuated microorganism is one which is still living, and capable of multiplying in the body after administration, but which has either been modified in some way to render it avirulent, or else is a strain which is virulent in other microorganisms, but avirulent in man. The advantage obtained by using an attenuated organism is that the attenuation usually does not affect its antigenicity, thereby providing a more efficient stimulus to the immune system. Attenuated vaccines for measles, rubella and poliomyelitis (Sabin vaccine) have achieved widespread use. Attenuation is typically achieved by altering the growth conditions of the microorganism, or, more recently, genetically modifying the organism""s virulence. Although generally more effective than killed vaccines, however, there is danger involved in the possibility of the live organisms reverting to virulence, thereby causing disease symptoms.
Because of the problems associated with whole organism vaccines, it has become more common in recent years to employ individual protective antigens as the active agent in vaccine compositions. Although isolation and identification of the particular antigens of an organism which do stimulate a protective response is not always simple, once identified, this method provides an effective alternative to the whole organism vaccines, without the attendant disadvantages.
Examples of types of antigens which are typically useful for this purpose are purified cell or capsid components, such as bacterial toxins (which must be detoxified to yield a toxoid), bacterial or viral toxin subunits, cell wall polysaccharides, or capsid glycoproteins. These components are often highly effective in producing immunity, but difficulties arise in the actual purification. It is critical that the antigen of interest be isolated more or less completely from extraneous cellular materials, the presence of which can frequently cause an adverse immunologic or metabolic response concurrent with the desired immunity producing reaction. The purification processes required are frequently complex, tedious and prohibitively expensive and the results not always completely predictable. This problem can in some case be avoided by synthetic preparation of small peptide sequences which correspond to epitopes on a microbial antigen. There are, in some circumstances, drawbacks to this technique in that, although the amino acid sequence corresponds to the natural epitope sequence, the conformation may not be sufficiently similar to that of the parent antigen to elicit the appropriate immune response.
Many of the disadvantages attendant upon these aforementioned vaccine types may be avoided by the use of recombinant DNA technology. The possibility of cloning genes which code for all or part of an important microbial antigen provides a relatively economical and convenient source of larger amounts of the necessary immunogenic material, substantially free of contaminating proteinaceous or genetic material. In brief, this method of producing purified antigens involves the insertion of the specific DNA sequence coding for the antigen into a DNA vector to form a recombinant DNA molecule which can be replicated by a host cell. There is now an abundance of methods by which recombinant DNA molecules may be prepared. One of the better known techniques is that described by Cohen and Boyer in U.S. Pat. No. 4,237,224, the teachings of which are herein incorporated by reference. In this method recombinant plasmids are produced using restriction enzymes and ligation; the resulting plasmids are then placed into a unicellular host cell which is thereby transformed, and begins replication of the foreign DNA. Another method, utilizing bacteriophage vectors, is described in U.S. Pat. No. 4,304,863, also incorporated herein by reference. The cloning method provides the greatest potential for making convenient, economical sources for vaccine preparation readily available; it is not without difficulties, however, in that the appropriate gene for an antigen known to be immunogenic must first be isolated and sequenced, inserted into a plasmid in such a way as to insure proper replication, transcription and translation, and then inserted into a compatible host cell.
The potential for failure of the expression of the desired gene product is tremendous if any one of transcription of the gene, translation of the RNA, or post-translational processing and compartmentalization of the polypeptide is not performed correctly. For a cloned insert to be transcribed, it is necessary that there is present a promoter which the host RNA polymerase can recognize. Proper translation requires that the RNA has a ribosome binding site. Post-translational modification of proteins often involves cleaving of a signal sequence which functions by directing the protein out through the cell membrane. Degradation of the foreign protein produced by the host microorganism may also occur if the configuration or amino acid sequence does not protect them from the action of the host""s intracellular proteases. Thus, although conceivably the ideal way of ultimately constructing a Neisseria vaccine, these techniques have not previously been applied to vaccine preparation for gonorrhea.
2.2. Gonococcal Antigens
Some antigens of Neisseria gonorrhoeae have been extensively studied and classified. Gonococcal pili, for example, have been found to be antigenic (Buchanan et. al., J. Clin. Invest. 52:2896-2909, 1973), consisting primarily of a protein subunit of about 20,000 M.W. A synthetic form of this material has been formulated into a vaccine preparation (Schoolnik et al., Prog. Allergy 33:314-331, 1983). The lipopolysaccharide of N. gonorrhoeae is also antigenic, with the xcex2-1, 4-linked galactose-glucose residues being the principal determinant. The greatest concentration of study in this area in recent years, however, has probably been focused on the outer membrane proteins, a complex of proteins whose role in immunogenicity is not yet fully understood. The composition of the outer membrane protein is known to vary somewhat from strain to strain, and on this basis at least 16 different serotypes have been identified (Johnston et. al., J. Exp. Med. 143:741-758, 1976). A number of vaccine compositions employing portions of the gonococcal membrane have previously been described, for example, in U.S. Pat. Nos. 4,203,971, 4,288,557 and 4,681,761. Most of these compositions, however, contain a mixture of protein and other material, and all require fairly elaborate purification procedures for isolation and separation of the active components from the bacterial cell. Thus, these vaccines may not have the desired immunospecificity, and also require a tremendous amount of microbial source material in order to produce commercial quantities of the product.
2.2.1. Gonococcal Protein I
Also suggested as a possible candidate for the basis of a vaccine composition is the specific outer membrane protein known as Protein I, or PI. PI is the major outer membrane protein of N. gonorrhoeae, functioning as a porin (Douglas et al., FEMS Microbiology Letters 12:305-309, 1981), a protein which is believed to operate in the cell by channelling low molecular weight substances across the hydrophobic lipid outer membrane. There are a number of features which make PI an interesting focus of attention: First, it is at least partly responsible for serotype specificity in Neisseria, and there are a relatively small number of antigenic serotypes of Protein I. Also, a number of gonococci possessing particular PI serotypes have been associated with complicated gonococcal infections. Further, it appears to be surface exposed in its native state, and also appears to stimulate the production of opsonins (Sarafian et al., J. Infect. Dis. 148:1025-1032, 1983). opsonins are antibodies which bind to the surface of an infectious organism, facilitating the engulfment of the organism by phagocytes. Its immunogenic potential has already been demonstrated in vaccinated mice (Jiskoot et al., Infect. Immun. 54:333-338, 1986).
Two different major types of PI molecules have been demonstrated in gonococci, PIA and PIB (Barrera et al., Infect. Immun. 44:565-568, 1984), based on peptide mapping and susceptibility to proteolysis (Blake et al., Infect. Immun. 33:212-222, 1981). This division has been found to correlate with serogroup patterns (Sandstrum et al., Infect. Immun. 35:229-239, 1982; Sandstrum et al., Infect. Immun. 38:462-470, 1982) and pathogenesis. Gonococci expressing protein IA are associated with systemic infections, while those with protein IB are associated with localized infection (Buchanan et al., Infect. Immun. 32:985-994, 1981; Hildebrandt et al., Infect. Immun. 20:267-273, 1978).
Despite all the attention paid to developing PI as a potential vaccine candidate, there has not yet been produced an effective vaccine based on PI. Further, there has not yet been any elucidation of the gene sequence controlling its production, and little is known about the protein""s structure. The present invention provides the first description of a full PI gene sequence, that of the PIA structural gene, as well as a cloned gene product. Also described is a novel PIB gene sequence, as well as unique PIA-PIB chimeras derived from these sequences. The latter chimeras are useful in epitope mapping as well as being a basis for vaccine development.
2.3. Diagnostic Probes
Another aspect of the widespread nature of this disease is the importance of early detection and diagnosis. There are, of course, standard bacteriological tests available for diagnosis of gonorrhea, but such tests rely on the growth of bacteria in culture which can be time consuming, and which is also generally rather non-specific. Neisseria gonorrhoeae is known to have different serotypes, which may be indicative of very distinct patterns of the disease, as noted in the previous section. In order to effect the appropriate treatment for the disease, it is critical that diagnosis not only be rapid, but also as accurate as possible.
The development of DNA- and RNA-probe technology has provided a solution to many such problems in diagnosis. A probe is typically a radiolabelled single strand of DNA or RNA which is complementary to all or a portion of a particular gene of interest, and therefore, when exposed to a single strand of the complementary nucleic acid, will hybridize to it. Probes are employed in a technique known as Southern blotting, in which DNA fragments from a sample suspected of containing the gene of interest are separated in agarose gels, denatured to create single strands, and then transferred to nitrocellulose filters. Here they are incubated with labelled, pre-selected probes, which will hybridize to a complementary strand and thus identify, by its label, the presence of the desired gene. Such probes can also be usefully employed in the identification of a particular clone containing a gene of interest from a genomic library established by cloning DNA fragments of an entire genome in host cells.
There has not heretofore been a convenient and highly accurate probe system developed in connection with Neisseria gonorrhoeae. The present invention, however, provides the first development of several oligonucleotide probes for N. gonorrhoeae, certain of which will hybridize generally to Protein I of N. gonorrhoeae but not to other bacteria, and others which are specific for N. gonorrhoeae serotypes which express the PIA antigen only. Thus is provided a reliable diagnostic method for detecting gonococcal infection, as well as identification of the particular category of serotype in which an individual may be infected.
The complete nucleotide sequence for the Protein IA, and for Protein IB, of Neisseria gonorrhoeae is described, as well as their predicted amino acid sequences. Also described are methods and compositions for the cloning and expression of the PIA and PIB gene in a single cell host organism, as well as cloning and expression of PIA-PIB chimeric protein-products. Also described are methods for culturing the novel host organism so as to produce the gene products. The products of the recombinant DNA methods employed are suitable for use in whole or in antigenic part, as immunogens in vaccine compositions to prevent gonorrhea.
The PIA and PIB genes were isolated from the bacterial genome by hybridization of DNA fragments with novel oligonucleotide probes. Once localized in a specific segment of DNA, the nucleotide sequence was determined and the amino acid sequence was predicted. Each gene was then inserted into a plasmid cloning vector which functions as the unit of replication of the gene. The recombinant plasmid was then used to transform a compatible host cell, whereby the gene product is expressed. Methods are also described herein which permit the isolation of the expressed products and the formulation of each into a vaccine composition. In particular, vaccine compositions comprising a hybrid PIA/PIB protein are also proposed. Based on the determination of nucleotide sequences of the PIA and PIB gene, the invention also provides the sequence of oligonucleotide probes which are useful in the detection and diagnosis of gonococcal infection. In this regard, the invention also contemplates diagnostic test kits comprising one or more of the oligonucleotide probes disclosed herein.